Transgenic plant production generally uses a single promoter sequence and a single 3′ untranslated region (UTR) to express a gene of interest in a transgenic plant. For example, a typical expression vector useful for expression of a gene of interest in maize includes a rice actin promoter and a 3′ untranslated region of a potato protease inhibitor II gene (PinII 3′ UTR, An et al. (1989) Plant Cell, 1: 115-122), flanking an open reading frame encoding the gene of interest. This method has been used to generate many transgenic events for different genes of interest, wherein all transgenic events share a common 3′ UTR (see, for example, U.S. Patent Application Publications 2003/0233670 and 2005/0108791, which are incorporated by reference). In this example, each gene of interest is expected to be constitutively expressed under the control of the rice actin promoter. Where an altered or specific expression pattern of the gene of interest is desired, this is generally accomplished by placing the gene of interest under the control of a different promoter (e. g., a tissue specific or temporal specific promoter), and producing additional transgenic events. These additional differentially regulated events allow the researcher to, e. g., better understand gene function or to associate specific traits with the particular expression of the transgene. Unfortunately, the re-cloning and production of additional transgenic events is costly and time consuming.
The present invention provides novel methods for rapidly and conveniently assessing the effects of different promoters on the expression of a given gene of interest or target RNA. The methods are compatible with investigating transgenic events that already exist (for example, the large sets of transgenic plants provided in U.S. Patent Application Publications 2003/0233670 and 2005/0108791). An expression-specific promoter operably linked to DNA for suppressing expression of a target RNA or gene of interest is used to silence the expression of the target RNA in a plant. In one embodiment, the method is done through genetic crosses of a first parent plant (or set of first parent plants) containing a target RNA (e. g., one or more coding sequences that all use a common 3′ UTR sequence that serves as the target RNA), with a second parent plant (or set of second parent plants), wherein each second parent plant has in its genome an expression-specific promoter operably linked to DNA for suppressing expression of the target RNA, and wherein the expression-specific promoter is different for each second parent plant. Preferably, such crosses produce hybrid progeny with selective expression of the target RNA relative to expression in the at least one first parent plant. The DNA for suppressing expression of the target RNA can be any DNA for gene suppression, including DNA that transcribes to single-stranded RNA or to double-stranded RNA or to both.
The invention provides rapid and convenient methods to alter, in a single generation, transgene expression patterns in a predetermined and rational manner. The methods are especially useful to allow the determination of tissues where expression of a gene of interest is desirable, e. g., in mode of action studies to determine where expression of a gene of interest is necessary or sufficient for activity. In a related utility, when creating transgenic plants for the purpose of screening genes for desired phenotypic effects, one may have only one or a few transgenic events displaying the desired phenotype; the invention provides methods by which one can confirm the function of a transgene by altering expression of the transgene in a given event. The method can also be used to optimize plant breeding strategies by allowing the alleviation of off types.